Severe Acute Respiratory Syndrome (“SARS”) is a human respiratory disease of recent origin, widespread infectivity, recurring incidence, and significant mortality. Specifically, SARS is a recently-observed human disease, with the first cases seen in Guangdong Province, China, in November, 2002. During this 2002-2003 outbreak, the World Health Organization (“WHO”) reported more than 30 countries in which the disease had occurred, with 774 of the 8096 patients who had contracted SARS eventually dying of the disease (see the WHO website at who.int/csr/sars/country/table2004—04—21/en/). Moreover, a second outbreak of SARS in four patients in the city of Guangzhou, Guangdong Province, China, between December, 2003, and January, 2004, demonstrated that the disease is recurrent, and therefore continues to be of serious impact to worldwide human health (see the WHO website at who.int/csr/don/2004—01—27/en/ and who.int/csr/don/2004—01—31/en/).
Subsequent to the initial SARS outbreak, an intensive collaborative research effort by the international scientific community identified the etiological agent causing the disease to be a novel coronavirus, the SARS coronavirus (“SARS-CoV” or, synonymously, “SCoV” or “SARS virus”) (Ksiazek et al., N. Engl. J. Med. 348:1947 (2003); Peiris et al., Lancet 361:1319 (2003); Drosten et al., N. Engl. J. Med. 348:1967 (2003)). This identification of the causative agent of SARS as a coronavirus is consistent with the known role of these viruses in animal and human respiratory diseases; as many as one third of all human mild upper respiratory tract illnesses, for example, are caused by human coronaviruses. Interestingly, however, although SARS-CoV is clearly a member of this diverse group of positive-stranded RNA viruses, based on RNA sequence comparisons it appears that SARS-CoV does not fall within any of the coronavirus evolutionary groups previously characterized, i.e., is not closely related to any previously known coronavirus (Rota et al., Science 300:1394 (2003); Marra et al., Science 300:1399 (2003)).
Although the agent responsible for SARS has been identified, successful prevention and treatment of the disease requires an additional understanding of the origin of SARS-CoV in humans, as well as knowledge of how the virus mutates during an outbreak of SARS. With regard to origin, as discussed above, SARS has only recently been observed in humans, suggesting the prior existence of SARS-CoV, or a close relative of SARS-CoV, in a separate, non-human source with which humans have had recent contact. Thus an important component in the control and prevention of future SARS outbreaks will be an understanding of this origin, including: knowledge of how the SARS coronavirus crosses this species barrier, i.e., the characteristics of the virus at the point when it first infects humans; and, an understanding of the non-human source of the coronavirus.
In this latter regard, a variety of data strongly implicate Himalayan palm civets (Paguma larvata; “palm civets” or, synonymously, “civets”) as the non-human source of SARS-CoV (although probably not the ultimate repository of the disease itself; see Example 2 below). First, the early cases of SARS in both the 2002-2003 and 2003-2004 outbreaks were associated with patient exposure to these exotic food animals, suggesting that they are the vectors for transmission to humans of SARS-CoV, or a close relative of SARS-CoV. And, second, it has been shown that palm civets indeed harbor a SARS-CoV-like coronavirus (synonymously “SCoV-like coronavirus”) highly related to SARS-CoV (99.8% RNA sequence homology), further suggesting the origin of the latter human form of the coronavirus from transmission of the former palm civet form (Guan et al., Science 302:276 (2003)). Despite this knowledge of the likely non-human source of the SARS coronavirus, however, the exact form of the virus at or immediately after transmission has not yet been elucidated.
With regard to the mutation of SARS-CoV during a SARS outbreak, a number of studies have demonstrated a variety of mutational changes in the SARS-CoV RNA sequences of various patients from the 2002-2003 outbreak (Ruan et al., Lancet 361:1779 (2003); Lan-Dian et al., Acta Pharmacol. Sin. 24:741 (2003)). Such changes are hardly surprising, in light of the recent introduction of SARS into humans from palm civets or some other non-human source discussed above, the strong selection pressures on the virus resulting after such a change in host, and the inherently high rate of genetic mutation in the coronaviruses resulting from their use of RNA as their genetic material. Such mutations in the underlying RNA genetic material are expected to result in new SARS coronaviral strains better adapted for growth in the human host, for ability to evade the human immune system, or with other novel properties which impact human health, for example the human-human hyper-infectivity of particular strains of the SARS coronavirus in what are termed “superspreader” events. Therefore, understanding the changes that occur in the SARS coronavirus during the course of an outbreak is critical to controlling and ultimately preventing the disease.
In light of the preceding discussion, it is clear that, although changes in the SARS coronavirus occurring at all stages of a SARS outbreak are important to an understanding of how to combat SARS, it is particularly important to understand the evolution of the SARS coronavirus in the earliest stages of its infection of humans, i.e., those stages at or immediately following the point at which the coronavirus crosses the species barrier. Such understanding of the earliest strains of the SARS coronavirus can be expected to lead to a variety of insights into prevention and treatment of the disease, including, for example: the development of molecular markers for identifying different evolutionary stages of the SARS-CoV (i.e., different stages occurring during a SARS outbreak), thereby allowing for the prediction of the severity of the disease in an infected patient, as well as likelihood of infectivity to others; the development of procedures based on the properties of the early SARS-CoV strains obtained, e.g., the use of the RNA genetic material of early SARS strains to obtain SARS coronaviral proteins important for the spread of these early strains (or their initial transmission to humans) for study and ultimately for targeting for drug inhibition; and, the use of these early SARS coronavirus strains in whole or in part in the development of vaccines to prevent SARS.
Despite this need for an understanding of the earliest stages of the SARS virus in humans, to date the data on the evolution of the SARS coronavirus during these earliest outbreak stages are limited. For example, Ruan et al. (Lancet 361:1779 (2003)) compared the RNA nucleotide sequences of fourteen SARS-CoV sequences from the 2002-2003 outbreak, only one of which (GZ01, also referred to in the literature as GD01) dates to the early stages of this outbreak. Although these data allowed Ruan et al. to make a number of statements regarding nucleotide positions of the SARS-CoV RNA associated with different regional SARS outbreaks, in light of the paucity of data from the earliest stages of the 2002-2003 outbreak, few conclusions can be drawn from the data of Ruan et al. about the critical earliest stages of the evolution of SARS-CoV.
There is thus a great need to obtain data regarding the earliest stages of the evolution of the SARS coronavirus in humans in order to understand, treat, and prevent SARS.